CD147 shRNA真核表达质粒的构建及鉴定

Construction of eukaryotic vector expressing shRNA of ovarian cancer associated antigen CD147

目的 针对CD147基因的不同部位,构建不同CD147shRNA表达质粒载体,在转录后水平抑制CD147的表达,对其克隆进行鉴定并挑选出抑制效率最高的克隆。方法 用DNA重组技术将针对人CD147基因的不同部位所设计的3对shRNA序列克隆到真核表达质粒pGE 1中,构建CD147shRNA表达质粒载体pGE 1CD147shRNA1、2、3。用阳离子脂质体介导转染人卵巢癌高转移细胞系HO 8910pm,经G418筛选后获得克隆进行鉴定。结果 3个CD147 shRNA表达载体pGE 1CD147shRNA1、2、3经PCR、限制性酶切及部分序列分析证明基因插入正确。半定量RT PCR、Westernblotting均证实:转染细胞8910pm/pGE 1CD147shRNA,与亲本细胞相比,CD147的表达在mRNA和蛋白水平均明显下降。结论 成功构建了CD147shRNA表达载体pGE 1CD147shRNA,并筛选出特异而高效地阻断CD147表达的克隆。此实验结果为进一步研究CD147蛋白肿拥纳镅Чδ芗坝τ玫於嘶?并对shRNA设计靶序列的选择有一定指导意义。

Objective To construct eukaryotic vector expressing shRNA (short hairpin RNA) of CD147. Methords Designing three different shRNA targeting the coding sequence of the CD147, the pGE-CD147 shRNA was constructed by inserting the designed shRNA to the eukaryotic expression vector pGE-1.The human ovarian cancer cell strain HO-8910pm was transfected by pGE-CD147 shRNA. After selected by G418,the CD147 expression in the transfected 8910pm cells was detected by RT-PCR and Western blot.Results It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vector expressing shRNA of CD147 was correct.The CD147 expression was significantly suppressed in 8910pm cells transfected by pGE-CD147 shRNA as compared with untransfected 8910pm cells. Conclusion The results of the study lay the foundation for further studying on biological functions and potential application of CD147 and give some guidance for the shRNA design.